Inactivation of Cryptosporidium parvum oocysts and Clostridium perfringens spores by a mixed-oxidant disinfectant and by free chlorine.

Cryptosporidium parvum oocysts and Clostridium perfringens spores are very resistant to chlorine and other drinking-water disinfectants. Clostridium perfringens spores have been suggested as a surrogate indicator of disinfectant activity against Cryptosporidium parvum and other hardy pathogens in water. In this study, an alternative disinfectant system consisting of an electrochemically produced mixed-oxidant solution (MIOX; LATA Inc.) was evaluated for inactivation of both Cryptosporidium parvum oocysts and Clostridium perfringens spores. The disinfection efficacy of the mixed-oxidant solution was compared to that of free chlorine on the basis of equal weight per volume concentrations of total oxidants. Batch inactivation experiments were done on purified oocysts and spores in buffered, oxidant demand-free water at pH 7 an 25 degrees C by using a disinfectant dose of 5 mg/liter and contact times of up to 24 h. The mixed-oxidant solution was considerably more effective than free chlorine in activating both microorganisms. A 5-mg/liter dose of mixed oxidants produced a > 3-log10-unit (> 99.9%) inactivation of Cryptosporidium parvum oocysts and Clostridium perfringens spores in 4 h. Free chlorine produce no measurable inactivation of Cryptosporidium parvum oocysts by 4 or 24 h, although Clostridium perfringens spores were inactivated by 1.4 log10 units after 4 h. The on-site generation of mixed oxidants may be a practical and cost-effective system of drinking water disinfection protecting against even the most resistant pathogens, including Cryptosporidium oocysts.     

Lethal effects of high-intensity violet 405-nm light on Saccharomyces cerevisiae,Candida albicans,and on dormant and germinating spores of Aspergillus niger

This study assessed the effects of high-intensity violet light on selected yeast and mould fungi. Cell suspensions of Saccharomyces cerevisiae, Candida albicans, and dormant and germinating spores (conidia) of the mould Aspergillus niger were exposed to high-intensity narrow band violet light with peak output at 405 nm generated from a light-emitting diode (LED) array. All three fungal species were inactivated by the 405-nm light without a requirement for addition of exogenous photosensitiser chemicals. Of the fungal species tested, S. cerevisiae was most sensitive and dormant conidia of A. niger were most resistant to 405-nm light exposure. Five-log₁₀ colony forming units per millilitre (CFU ml^?1) reductions of the tested species required exposure doses of 288 J cm^?2 for S. cerevisiae, 576 J cm^?2 for C. albicans, and a much higher value of 2.3 kJ cm^?2 for dormant conidia of A. niger. During germination, A. niger conidia became more sensitive to 405-nm light exposure and sensitivity increased as germination progressed over an 8 h test period. Light exposure under aerobic and anaerobic conditions, together with results obtained using ascorbic acid as a scavenger of reactive oxygen species, revealed that 405-nm light inactivation in fungi involved an oxygen-dependent mechanism, as previously described in bacteria. The inactivation results achieved with yeast cells and fungal spores together with operational advantages associated with the use of a visible (nonultraviolet (UV)) light source highlight the potential of 405-nm light for fungal decontamination applications.     

Effectiveness of Disinfectants in Killing Enterobacter sakazakii in Suspension, Dried on the Surface of Stainless Steel, and in a Biofilm

The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.     

Susceptibility of Clostridium Sporogenes Spores to Selected Reference Substances and Disinfectants

Research on the susceptibility of the spores of anaerobic bacteria such as Clostridium sporogenes or Clostridioides difficile is vital for assessing the sporicidal activity of disinfectants. The diverse susceptibility of anaerobic bacteria spores may lead to different disinfection parameters being determined by laboratories that prepare spore suspensions to test sporicidal effectiveness. The tests were performed using the suspension method according to PN-EN 13704:2018-09. In order to assess the susceptibility of the C. sporogenes spores, the criterion established for the C. difficile ribotype 027 spores was used in accordance with PN‑EN 17126:2019-01. The susceptibility of the C. sporogenes spores to glutardialdehyde corresponded to the susceptibility ranges established for the C. difficile ribotype 027 spores. The C. sporogenes spore suspension was susceptible to low concentrations of peracetic acid (0.01%). A disinfectant containing peracetic acid as the active substance showed high sporicidal activity at a low concentration (1%), a short contact time (15 minutes), and a high organic load (3.0 g/l bovine albumin + 3.0 ml/l sheep erythrocytes), as compared to a disinfectant with glutardialdehyde, which was sporicidal at a higher concentration (2.5%), at a longer contact time(60 minutes) and lower organic conditions (3.0 g/l bovine albumin). There is a need to define the minimum susceptibility criteria for the C. sporogenes spores to the reference substances most often found in disinfectants with sporicidal activity. Excessive susceptibility of the C. sporogenes spores to reference substances may result in low-performance parameters of disinfection products with sporicidal activity and lead to ineffective disinfection in practice. Open in a separate window     

Sterilization of biological pathogens using supercritical fluid carbon dioxide containing water and hydrogen peroxide

Novel noninvasive techniques for the removal of biological contaminants to generate clean or sterile materials are in demand by the medical, pharmaceutical and food industries. The sterilization method described here uses supercritical fluid carbon dioxide (SF-CO₂) containing 3.3% water and 0.1% hydrogen peroxide (v/v/v) to achieve from four to eight log viability reduction of all tested microbial species, including vegetative cells, spores and biofilms. The sterilization method employs moderate pressure and temperature (80 atm, 50 °C) and a short (30-minute) treatment time. The procedure kills various opportunistic pathogens that often persist in biofilm structures, fungal spores commonly associated with nosocomial infections, and Bacillus pumilus SAFR-032 endospores that are notoriously hard to eradicate by conventional sterilization techniques.     

Menacing Mold: Recent Advances in Aspergillus Pathogenesis and Host Defense

The genus Aspergillus is ubiquitous in the environment and contains a number of species, primarily A. fumigatus, that cause mold-associated disease in humans. Humans inhale several hundred to several thousand Aspergillus conidia (i.e., vegetative spores) daily and typically clear these in an asymptomatic manner. In immunocompromised individuals, Aspergillus conidia can germinate into tissue-invasive hyphae, disseminate, and cause invasive aspergillosis. In this review, we first discuss novel concepts in host defense against Aspergillus infections and emphasize new insights in fungal recognition and signaling, innate immune activation, and fungal killing. Second, the review focuses on novel concepts of Aspergillus pathogenesis and highlights emerging knowledge regarding fungal strain heterogeneity, stress responses, and metabolic adaptations on infectious outcomes. Mechanistic insight into the host–pathogen interplay is thus critical to define novel druggable fungal targets and to exploit novel immune-based strategies to improve clinical outcomes associated with aspergillosis in vulnerable patient populations.     

Residue aspects of disinfectants used in the food industry

Disinfectants are frequently used in the food industry against harmful (micro-)organisms on equipment (surfaces, pipelines etc.) which can come into contact with food. In The Netherlands, such uses of disinfectants are only allowed when registered according to the provisions of our national Pesticide Law. In most cases, (registered) uses require an operating procedure consisting of 3 consecutive steps: cleaning, disinfection and rinsing the equipment with clean water, thus avoiding contamination of food as much as possible. However, in some cases traces of the used compound(s) in food are inevitable. Therefore, Maximum Residue Limits (MRLs) for the disinfectants involved are established in food in the Netherlands. As remaining residues must be safe when ingested by the public, intake must be well below the Acceptable Daily Intake (ADI) for the respective chemicals. Also, sensitive and easy-to-operate methods of residue analysis, capable of detecting low levels of the compounds in food, must be available for enforcement purposes. In the Netherlands, experience with the existing legislation was gained over the past 25 years, resulting in the establishment of some 15–20 MRLs for residues of disinfectants in food.Harmonisation of the use of disinfectants (and other biocides) in the EU will be achieved in a draft ‘Biocide Directive’ in which the residue aspects of disinfectants are also covered, in a more-or-less comparable way as has been layed down in the Dutch legislation.Recent developments include the use of disinfectants in food as such, to reduce micro-organisms and to prolong the shelf-life of food products. Although in some surrounding countries this technique may already have become common practice. The Netherlands is reluctant to register those uses, and restricts it to cases where there is an inevitable technical need for disinfection, to minimize the exposure of the consumer to residues of (potentially) dangerous chemicals.     

New Formaldehyde Base Disinfectants

Preparations of formaldehyde in various organic liquids-ethylene glycol, glycerol, and propylene glycol-serve as effective disinfectants towards microbial vegetative cells and spores. This disinfection is a temperature-dependent process and is manifest when these formaldehyde base disinfectants are dissolved in water. The irritating vapors associated with formaldehyde disinfection are not present in either of these new formaldehyde base disinfectants or in aqueous solutions of them.     

Bacterial disinfectant resistance—a challenge for the food industry

The focus on hygiene in the food industry has resulted in an increasing use of chemical disinfection and it has been speculated that this will impose a selective pressure and contribute to the emergence of disinfectant-resistant microorganisms. The frequency of strains with a low-level resistance to quaternary ammonium compounds (QAC) is relatively high for Listeria monocytogenes (10%), Staphylococcus spp. (13%) and Pseudomonas spp. (30%) and lower for lactic acid bacteria (1.5%) and coliforms (1%) isolated from food and food processing industry. In general, bacteria isolated after disinfection are more resistant and represent a potential food safety or food spoilage problem. Adaptation to disinfectants may be accompanied by cross-resistance to related disinfectants. We have recently found a genetic linkage between resistance to QAC and antibiotics in food associated staphylococci, and there is a growing concern about cross-resistance between antibiotics and disinfectants.Disinfectant resistance can in most cases be prevented by effective cleaning and disinfection procedures. More effort should be made to avoid build-up of resistance in food production environments.     

Developmental regulators in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

The filamentous fungus Aspergillus fumigatus is the most prevalent airborne fungal pathogen causing severe and usually fatal invasive aspergillosis in immunocompromised patients. This fungus produces a large number of small hydrophobic asexual spores called conidia as the primary means of reproduction, cell survival, propagation, and infectivity. The initiation, progression, and completion of asexual development (conidiation) is controlled by various regulators that govern expression of thousands of genes associated with formation of the asexual developmental structure conidiophore, and biogenesis of conidia. In this review, we summarize key regulators that directly or indirectly govern conidiation in this important pathogenic fungus. Better understanding these developmental regulators may provide insights into the improvement in controlling both beneficial and detrimental aspects of various Aspergillus species.     

Effect of bongkrekic acid on growth and metabolism of filamentous fungi

The antifungal activity of bongkrekic acid against 17 tested molds was determined. Bongkrekic acid prevented spore germination and mycelial proliferation of Aspergillus niger, Rhizopus oryzae and Penicillium italicum. The action of bongkrekic acid was fungicidal. Under these conditions, the incorporation of ¹⁴C-leucine and ¹⁴C-uracil into the perchloric acid insoluble material of germinating A. niger conidia was significantly reduced by bongkrekic acid. Respiratory activity of resting spores was not affected by bongkrekic acid. Respiratory activity of germinated spores was inhibited by bongkrekic acid to the extent of 30 to 60% of controls for A. niger, R. oryzae and P. italicum. It has been concluded that operation of adenine nucleotide translocation in mitochondria of tested fungi is obligatory both for normal spore germination and fungal growth.     

Sporicidal effect of disinfectants on Bacillus cereus isolated from the milk processing environment

The sporicidal efficacy of sodium hypochlorite and a combination of peracetic acid and hydrogen peroxide on Bacillus cereus spores isolated from the milk processing environment was examined using the European Suspension Test and by a surface disinfection test on stainless steel and rubber. The results of the laboratory tests were compared with field trials in a milking installation. In general, it was difficult to obtain consistent results, as the repeatability and reproducibility of the tests were found to vary according to the test strain, spore suspension preparation, disinfectant test solution, organic load, contact time and temperature. The sporulation medium used to obtain spores influenced the sporicidal effect considerably. To overcome this problem a standard method for preparation of spore suspensions should be prescribed. The various disinfectants were more effective in suspensions than on surfaces and in field trials. For the suspension tests SE values ranging from 1.0 to 3.0 were reached within 10 min at 50°C, depending on the disinfectant used. Sodium hypochlorite-based products were most effective. The activity on spores on surfaces and in field trials was limited. In surface tests reductions of 0.4–0.8 were observed within 10 min at 50°C depending on the type of surface. The SE values obtained for rubber were lower compared with stainless steel. The decrease in spore levels found in the milking installation was comparable with the surface experiments, i.e. 0.4–1.0. It is important to develop standard test procedures to assess the sporicidal efficacy of disinfectants used in food hygiene. Surface tests should be included to reflect the in-use conditions more closely and minimum standards should be determined for both suspension tests and surface tests.     

Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi Beauveria bassiana and Metarhizium anisopliae

The effects of nutrient medium composition and temperature on the germination of conidia of the fungi Beauveria bassiana (strain AlG) and Metarhizium anisopliae (strain M-99) and their entomopathogenic activity have been studied. It was demonstrated that the presence of carbohydrates alone was sufficient for the spores of M. anisopliae M-99 to germinate, whereas the germination of B. bassiana AlG spores was inhibited by carbohydrates. Addition of KJ, ZnSO₄, or KBr into the Czapek medium increased the entomopathogenic activity of B. bassiana. The optimum temperature for spore germination was 20–35°C in both fungal species.     

Phenylalanine ammonia-lyase activity in alfalfa suspension cultures treated with conidia and elicitors of Verticillium albo-atrum

Alfalfa (Medicago sativa L.) cell suspension cultures of Verticillium albo-atrum resistant and susceptible genotypes were established from leaf callus tissues. Treatment of cultures with conidia and heat-released elicitors of V. albo-atrum induced a large increase in the activity of phenylalanine ammonia-lyase, only in the cells of the resistant genotypes with a maximum after 12 h. In co-cultivation with the fungal conidia and resistant cell lines, the production of spores were inhibited. This revised version was published online in July 2006 with corrections to the Cover Date.     

Efficacy of some commerical disinfectants against the bacterial isolates from a poultry slaughterhouse in Turkey

In this study, efficacy of seven different commercial disinfectant preparations was investigated against characteristic bacteria of a poultry slaughterhouse in Ankara (Turkey) by using paper disc-agar diffusion method and surface effectiveness test. According to paper disc-agar diffusion method, some disinfectants had wide efficacy against the test bacteria. The disinfectant effectiveness generally increased by the increasing of disinfectant concentration. However, some disinfectants were ineffective even though at their highest concentrations. The most effective disinfectant was B which contains QAC as active agent.Staphylococcus spp.,Staphyloccus aureus andEscherichia coli were the most sensitive bacteria to the disinfectants. Some pathogenic isolates, especiallySalmonella spp. andCampylobacter spp., were the most resistant ones to many of the disinfectants tested. The results of paper disc-agar diffusion method indicated the importance of characteristic bacterial strains of food plants as the test bacteria for disinfectant efficacy tests. Conversely of paper disc-agar diffusion method, all disinfectants were effective against the isolates by surface effectiveness test depending on exposure time. The disinfectants, except A and F, produced at least 3 log unit reduction during 5 min of exposure. However, all disinfectants at their lowest concentrations were effective against all tested bacteria during 15 and 30 min by this test.     

Entomophthora brevinucleata sp. nov. [Zygomycetes,Entomophthoraceae], a pathogen of gall midges [Dip.: Cecidomyiidae]

Entomophthora brevinucleata, a fungal pathogen of gall midges, is described. The species occurs on at least 3 host species, including the wheat blossom midgeSitodiplosis mosellana Géhin andMycodiplosis sp.E. brevinucleata produces rhizoids, either monohyphal or comprising bundles of a few hyphae, without holdfasts. The hyphal bodies are subspherical with mean dimensions of 27.5×22.1 μm. The primary conidia are campanulate with mean dimensions from individual hosts of 11.2–18.9×8.7–15.8 μm. They contain 3–13 nuclei with a mean diameter of between 2.7 and 3.7 μm stained with lactophenol-aceto-orcein. The secondary conidia are of similar shape but smaller. Cystidia and resting spores were not observed.     

A Murine Inhalation Model to Characterize Pulmonary Exposure to Dry Aspergillus fumigatus Conidia

Most murine models of fungal exposure are based on the delivery of uncharacterized extracts or liquid conidia suspensions using aspiration or intranasal approaches. Studies that model exposure to dry fungal aerosols using whole body inhalation have only recently been described. In this study, we aimed to characterize pulmonary immune responses following repeated inhalation of conidia utilizing an acoustical generator to deliver dry fungal aerosols to mice housed in a nose only exposure chamber. Immunocompetent female BALB/cJ mice were exposed to conidia derived from Aspergillus fumigatus wild-type (WT) or a melanin-deficient (Δalb1) strain. Conidia were aerosolized and delivered to mice at an estimated deposition dose of 1×10⁵ twice a week for 4 weeks (8 total). Histopathological and immunological endpoints were assessed 4, 24, 48, and 72 hours after the final exposure. Histopathological analysis showed that conidia derived from both strains induced lung inflammation, especially at 24 and 48 hour time points. Immunological endpoints evaluated in bronchoalveolar lavage fluid (BALF) and the mediastinal lymph nodes showed that exposure to WT conidia led to elevated numbers of macrophages, granulocytes, and lymphocytes. Importantly, CD8⁺ IL17⁺ (Tc17) cells were significantly higher in BALF and positively correlated with germination of A. fumigatus WT spores. Germination was associated with specific IgG to intracellular proteins while Δalb1 spores elicited antibodies to cell wall hydrophobin. These data suggest that inhalation exposures may provide a more representative analysis of immune responses following exposures to environmentally and occupationally prevalent fungal contaminants.     

Bacterial Spores Survive Treatment with Commercial Sterilants and Disinfectants

This study compared the activity of commercial liquid sterilants and disinfectants on Bacillus subtilis spores deposited on three types of devices made of noncorrodible, corrodible, or polymeric material. Products like Renalin, Exspor, Wavicide-01, Cidexplus, and cupric ascorbate were tested under conditions specified for liquid sterilization. These products, at the shorter times indicated for disinfection, and popular disinfectants, like Clorox, Cavicide, and Lysol were also studied. Data obtained with a sensitive and quantitative test suggest that commercial liquid sterilants and disinfectants are less effective on contaminated surfaces than generally acknowledged.     

Comparative Effects of Osmotic, Sodium Nitrite-Induced, and pH-Induced Stress on Growth and Survival of Clostridium perfringens Type A Isolates Carrying Chromosomal or Plasmid-Borne Enterotoxin Genes

About 1 to 2% of Clostridium perfringens isolates carry the enterotoxin gene (cpe) necessary for causing C. perfringens type A food poisoning. While the cpe gene can be either chromosomal or plasmid borne, food poisoning isolates usually carry a chromosomal cpe gene. Previous studies have linked this association between chromosomal cpe isolates (i.e., C-cpe isolates) and food poisoning, at least in part, to both the spores and vegetative cells of C-cpe isolates being particularly resistant to high and low temperatures. The current study now reveals that the resistance phenotype of C-cpe isolates extends beyond temperature resistance to also include, for both vegetative cells and spores, enhanced resistance to osmotic stress (from NaCl) and nitrites. However, by omitting one outlier isolate, no significant differences in pH sensitivity were detected between the spores or vegetative cells of C-cpe isolates versus isolates carrying a plasmid-borne cpe gene. These results indicate that both vegetative cells and spores of C-cpe isolates are unusually resistant to several food preservation approaches in addition to temperature extremes. The broad-spectrum nature of the C-cpe resistance phenotype suggests these bacteria may employ multiple mechanisms to persist and grow in foods prior to their transmission to humans.     

Eicosanoid biosynthesis inhibitors influence mortality of Pieris brassicae larvae co-injected with fungal conidia

Influence of fungal species (conidia spores) on mortality of Pieris brassicae larvae differed when injected into the larvae. The effects of B. bassiana (ARSEF-1151) were expressed in a conidial dose-dependent manner on mortality of the larvae. An increased and faster mortality of the larvae followed B. bassiana (ARSEF-1151) infection when the spores were co-injected with the eicosanoid biosynthesis inhibitors (dexamethasone, naproxen, phenidone, esculetin). These compounds express different modes of action. These lethal effects were reversed when dexamethasone was injected with eicosanoid precursor arachidonic acid (20:4n-6). Nodulation is the predominant cellular reaction to bacterial and fungal injection in insects. Injection of each of five fungal species showed that nodulation intensity varies according to infecting fungal species. These findings support the idea that virulent effects of entomopathogenic, fungal species can be increased when P. brassicae immune systems are suppressed.